Technical Reference #212

212.

Phosphorylation-dependent Localization of Microtubule-associated Protein MAP2c to the Actin Cytoskeleton Rachel S. Ozer and Shelley Halpain, The Scripps Research Institute, Molecular Biology of the Cell, 11(212), (2000)
Link To Paper

Abstract:
Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubulegrowth and actin cross-linking and bundling in vitro. Little is known about MAP2regulation or its interaction with the cytoskeleton in vivo. Here we inve

Materials & Methods:
Plasmids encoding EGFP-MAP2c as a fusion protein were derived from the previously described pET3aMAP2c (Gamblin et al. 1996). The coding region for MAP2c was amplified by using primers that generate HinDIII and BamHI sites at the 59 and 39 ends respectively. The polymerase chain reaction product was ligated into HinDIII- and BamHI-digested pEGFP-C-3 vector (Clontech Palo Alto CA). Mutation of S319 S350 and/or S382 to alanine (A) or glutamic acid (E) was carried out by using the Quik-Change mutagenesis kit (Stratagene La Jolla CA) and all constructs were sequenced after mutagenesis. Plasmid DNA was prepared for transfection by transformation into Escherichia coli XL2 Blue cells and selection for kanamycin resistance. Plasmid DNA for transfections was isolated and purified by using the Qiafilter maxiprep kit (Qiagen Valencia CA). Recombinant Protein Expression Purification Phosphorylation and 2-Dimensional (2-D) Phosphopeptide Mapping Thermally stable MAP2c was isolated from E. coli BL21 (DE3) cells (Stratagene) as described previously (Gamblin et al. 1996) with the exception that NaCl in the bacterial lysate was reduced to 150 mM. This material was further purified by ion exchange chromatography by using a 1-ml HiTrap SP cation-exchange column (Pharmacia Piscataway NJ) equilibrated with 50 mM sodium acetate 1 mM MgCl2 1 mM EGTA and 1 mM dithiothreitol pH 5.5. Boiled and centrifuged bacterial lysate from 0.5–1 liter cultures containing 10–20 mg of MAP2c was loaded onto the column and the column was washed with .10 volumes of equilibration buffer with 0.15 M NaCl added. Purified MAP2c was eluted from the column by using equilibration buffer with the addition of 0.25 M NaCl. Fractions containing purified full-length MAP2c (.90%) were identified by SDS-PAGE and pooled. For in vitro phosphorylation by PKA MAP2c was buffer-exchanged by using a PD-10 gel filtration column (Pharmacia) into 50 mM HEPES pH 7.4 10 mM MgCl2 1 mM EDTA 1 mM EGTA 2 mM dithiothreitol and protein concentration was determined (Coomassie Protein Plus reagent; Pierce Rockford IL). Phosphorylation reactions were conducted at 5 mM MAP2c with 200 mM ATP (labeled for phosphopeptide mapping to 0.2– 0.4 Ci/mmol with [g-32P]ATP [6000 Ci/mmol; New England Nuclear Boston MA]) and purified PKA (Pierce) 2 u/mg MAP2c. Samples were preincubated at 30°C for 5 min and reactions initiated by the addition of ATP. After specific incubation times aliquots were withdrawn and reactions halted by addition to SDS-loading buffer (final concentration: 3% SDS 62 mM Tris 5% glycerol 20 ml/ml b-mercaptoethanol) and immediate boiling for 3 min. Stoichiometry was calculated by purification of the phosphorylation reaction by SDS-PAGE excision of the gel piece containing MAP2c and Cerenkov counting of phosphate incorporation. Phosphopeptide mapping (2-D) was performed as described (Hemmings et al. 1984) and evaluated via phosphorimager analysis by using ImageQuant software (Molecular Dynamics Sunnyvale CA). Native Protein Isolation Antibody Production and Matrix-assisted Laser Desorption Ionization (MALDI) Mass Spectrometry A new polyclonal antibody to MAP2c was developed by immunization of a New Zealand White rabbit with 400 mg of purified MAP2c in sterile phosphate-buffered saline (PBS) and complete Freund’s adjuvant. Serum was collected weekly beginning 4 wk after the initial injection. Additional injections were administered 7 wk after the initial injection and at 6-wk intervals thereafter containing 400 mg of MAP2c and incomplete Freund’s adjuvant. The serum was designated 4170. Rat hippocampal homogenate was prepared by dissecting a postnatal day 10 rat hippocampus sonication into 500 ml of boiling 1% SDS and boiling for 2 min. Protein content was assayed by using bicinchoninic acid protein assay reagent (Pierce). MAP2c was immunoprecipitated from postnatal day 10 hippocampal homogenate (200 mg) with 10 ml of 4170 serum as described previously (Halpain and Greengard 1990). Coomassie staining of 8% polyacrylamide gels indicated that high- and lowmolecular- weight MAP2s were the only proteins detectable in addition to immunoglobulin. The MAP2c gel piece was excised for mass spectrometry analysis affording a sample of .90% pure native MAP2c for analysis. Either recombinant or native MAP2c was prepared for MALDI mass spectrometry by dialysis of the MAP2c-containing gel piece against 2–3 liters of water for 3 h to remove SDS and salt and incubation overnight at 37°C in 300 ml of 25 mM NH4HCO3 with 0.02 mol/mol sequencing grade trypsin (Promega Madison WI). The supernatant was removed and the gel piece extracted with 60% acetonitrile 0.01% trifluoroacetic acid (33 250 ml for 20 min each) and all extracts were combined with the digest and evaporated to dryness. MALDI time-of-flight reflectron mass spectrometry was performed by the Scripps Research Institute Core Facility (more information available at http://masspec.scripps.edu/instr.descrip. html). Ions were identified by using a 0.1% margin of precision in the PAWS 3.0 software and were only assigned when there was a unique peptide within MAP2c matching the observed mass.

Microscopic Technique
Live Cell Microscopy, Confocal Microscopy, Deconvolution Microscopy

Cell Type(s)
HeLa